Project Summary/Abstract Endochondral ossification involves a highly coordinated program of chondrocyte differentiation, proliferation, maturation and hypertrophy. The cellular activities of chondrocytes are organized in distinct zones in the growth plate, making this tissue uniquely suited for in vivo studies, as effects of mutations can be linked to specific cellular activities. The bone morphogenetic protein (BMP) and transforming growth factor beta (TGFbeta)/activin pathways are important regulators of these processes. The importance of extracellular antagonists as regulators of the duration, intensity and extent of BMP and TGFbeta/activin signaling has been defined. For example, mice lacking the BMP receptor antagonist Noggin exhibit cartilage overgrowth concurrent with excess BMP activity. However, very little is known about the roles of intracellular inhibitors of BMP and TGFbeta/activin pathways, such as the inhibitory Smad (I-Smad) proteins, Smad6 and Smad7. Gain and loss of function studies have revealed potential roles for I-Smads in chondrocytes in vitro, but no in vivo studies have been performed. The long-term goal of this project is to understand how intracellular regulation of BMP and TGFbeta/activin signaling by I-Smads controls chondrogenesis as a prerequisite to more efficient strategies utilizing BMPs to achieve cartilage maintenance and repair. To achieve this goal, it is important to determine whether I-Smads are critical for chondrogenesis and to identify specific aspects of chondrogenesis, as well as mechanisms, impacted by I-Smads. Hence, the specific hypotheses to be addressed in this proposal are (1) I-Smads are essential for normal endochondral bone formation, (2) loss of I-Smads impacts the balance of signaling cross-talk between TGFbeta/activin and BMP pathways in the growth plate that will affect the progression of endochondral ossification, and (3) TGFbeta signaling interact via Smad7 with key survival/stress pathways in the growth plate.